Pathway Acetaminophen Pathway (therapeutic doses), Pharmacokinetics

Stylized diagram showing acetaminophen metabolism and transport in the liver.
Acetaminophen Pathway (therapeutic doses), Pharmacokinetics
phenobarbital phenytoin APAP isoniazid ethanol ugt1a9 ugt2b15 ugt1a6 ugt1a1 sult1e1 sult1a1 sult1a3 sult1a4 sult2a1 cyp3a4 cyp1a2 cyp2d6 cyp2a6 cyp2e1 gstp1 gstt1 gstm1 abcc3 abcc4 abcg2 abcc2
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Acetaminophen (N-acetyl-p-aminophenol, APAP, or paracetamol, PARA) is widely used for its analgesic and antipyretic properties in many over-the-counter formulations in both adults and children [Articles:21054454, 23719833]. APAP can be synthesized in the body through O-dealkylation of the prodrug phenacetin, a pain-killer that was withdrawn from the market due to nephrotoxicity and carcinogenesis [Article:7002186]. At the therapeutic adult dose of 1-2 g/day, oral APAP is indicated for fever and mild to moderate acute pain conditions [Article:23719833]. Administration of acetaminophen via intravenous route has become increasingly widespread and has been used as a safe and effective antipyretic and analgesic agent [Article:25521845]. Maximum recommended therapeutic dose of APAP is 4 g/day in adults and 50-75 mg/kg/day in children. Consumption of a single dose greater than 7 g in an adult and 150 mg/kg in a child is considered potentially toxic to the liver and kidneys due to the highly active metabolite N-acetyl-p-benzoquinone imine (NAPQI)[Article:22998987].


The liver, and to a lesser extent the kidney and intestine, are the major organs implicated in the metabolism of acetaminophen [Article:11215692]. After a therapeutic dose, APAP is mostly converted to pharmacologically inactive glucuronide (APAP-gluc, 52-57% of urinary metabolites) and sulfate (APAP sulfate, 30-44%) conjugates, with a minor fraction being oxidized to a reactive metabolite NAPQI (5-10%). Less than 5% of APAP is excreted unchanged [Article:6343056]. NAPQI is highly reactive and is primarily responsible for acetaminophen-induced hepatotoxicity. Detoxification of NAPQI occurs through its binding to the sulfhydryl group of glutathione (GSH) to form APAP-GSH, which is ultimately excreted in the urine as cysteine and mercapturic acid conjugates (APAP-cys) [Articles:22998987, 11215692]. Acetaminophen disposition involves a complex interorgan transport of metabolites between the liver, kidney and intestine, through bile and blood stream, to be ultimately eliminated in urine and feces [Article:11215692]. From the liver, most of glucuronide and sulfate metabolites get transported into the kidneys through the blood stream, while some APAP-gluc appears in the bile with subsequent transport into the intestine. The kidney is the main site of the disposition of APAP sulfate, either through direct excretion or through further biotransformation followed by renal excretion. Although most of NAPQI is formed in the liver, the kidney also metabolizes APAP to the toxic metabolite and releases cysteine conjugate of APAP into the bile and blood for further elimination in urine [Article:11215692].

At supratherapeutic doses of APAP (more than 4 g/day), sulfation pathway becomes saturated, while glucuronidation and oxidation increase, and a smaller amount is excreted unchanged. After a highly toxic dose of APAP, glucuronidation gets saturated as well and higher proportions of the drug are eliminated unchanged (~10%) and get oxidized to NAPQI (>15%) ( Excess NAPQI eventually depletes GSH stores and starts to form protein adducts through binding to cysteine groups on cellular proteins. NAPQI primarily targets mitochondrial proteins and ion channels leading to the loss of energy production, ion misbalance and cell death [Articles:22998987, 11215692, 14625346]. Following animal studies, N-acetylcysteine (NAC) was shown to be an effective antidote for acetaminophen overdose in humans [Article:7469630]. NAC replenishes GSH stores, scavenges reactive oxygen species in mitochondria and enhances the sulfation metabolic pathway ( If administered within 8-10 hours after an acute overdose, NAC reduces the risk of hepatotoxicity to less than 5%. Overall, NAC prevents liver damage, renal failure and death, and is the treatment of choice for APAP poisoning [Articles:22998987, 11215692, 6343056, 3059186]. Extremely high doses of APAP result in severe liver damage accompanied by dramatically diminished glucuronidation and sulfation capacities [Article:6343056]. In patients with fatal centrilobular hepatic necrosis, plasma and urinary levels of glucorinide metabolite are almost undetectable [Article:4788034].

Glucuronidation pathway of acetaminophen metabolism is catalyzed by UDP-glucuronosyl transferases (UGT). UGTs make APAP molecule more water-soluble by transferring the glucuronosyl group from UDP-glucuronic acid [Articles:22998987, 23462933]. Studies in human liver microsomes and cultured hepatocytes indicate that UGT1A1, UGT1A6, UGT1A9 and UGT2B15 are involved in APAP glucuronidation [Articles:8494539, 11714888, 16696573, 15933229]. UGT1A6 is important at low APAP concentrations [Article:11714888], while UGT1A9 and UGT1A1 contribute the most at toxic doses with UGT1A9 catalyzing within a broad range of pharmacologically relevant APAP concentrations [Articles:11714888, 16696573].

A family of cytosolic enzymes, called sulfotransferases (SULT), carries out sulfation of acetaminophen. SULTs transfer a sulfo group from a substrate PAPS to APAP making it more polar and prone to elimination [Article:23462933]. Using human platelet homogenates as a model for xenobiotic metabolism in the liver, SULT1A1 and SULT1A3/4 were first shown to catalyze APAP sulfation [Article:6950087]. Human SULT1A3 and SULT1A4 genes are very closely related and code for identical SULT proteins [Article:14676822]. In addition to SULT1A1 and 1A3/4, sulfation of APAP in the human fetal liver is carried out by SULT1E1 and SULT2A1 [Article:18232020]. This study showed that in the fetal liver, SULT1A3/4 plays the major role in APAP sulfation; in the postnatal development, however, APAP is predominantly sulfated by SULT1A1 and SULT2A1, while SULT1A3/4 activity diminishes [Article:18232020].

Cytochrome P450 enzymes catalyze oxidation of acetaminophen to the reactive metabolite NAPQI [Articles:23462933, 11215692]. The exact contribution of particular CYP isoforms to APAP bioactivation varies and depends on the concentration of the drug. In human liver microsomes, CYP2E1 and CYP1A2 were first reported to convert high doses of APAP to NAPQI [Article:2729995]. Later studies with purified human proteins or human liver microsomes and specific inhibitors confirm the role of CYP2E1 in bioactivation of toxic levels of APAP but also report involvement of CYP2A6 [Articles:9548799, 11866476]. Studies with healthy human volunteers pre-treated with CYP2E1 inhibitor disulfiram further confirm the role of CYP2E1 in APAP oxidation [Article:10741631]. Using human liver microsomes and human recombinant CYP2D6, this enzyme has been reported to oxidize only very high, toxic doses of APAP, when plasma APAP concentration reaches 2 mM [Articles:11095574, 19219744]. The role of CYP3A4 in APAP metabolism is controversial, with findings ranging from no significant contribution to the primary role in APAP oxidation [Articles:10741631, 19219744, 8387297, 8374050].

Conjugation of NAPQI to GSH occurs via both a spontaneous process and an enzymatic reaction catalyzed by glutathione-S-transferases (GSTs) [Article:3395122]. Nonenzymatic reaction yields a GSH conjugate, 3-(glutathione-S-yl)-acetaminophen (APAP-GSH); a reduction product, free APAP; and an oxidation product, glutathione disulfide (GSSG). GST reaction yields APAP-GSH and free APAP. The human cytosolic GST family is comprised of seven distinct classes of enzymes with numerous genetic variants within each class [Article:23201197]. Human in vitro studies with isolated liver and placenta GSTs have shown that GSTP1 is the most effective catalyst of NAPQI conjugation with GSH, followed by GSTT1 and GSTM1 [Article:3395122]. In the NAPQI reduction reaction, the most efficient human transferase is GSTT1, followed by GSTM1 and GSTP1.


Disposition and elimination of acetaminophen depend on its transport through different cell types. Unlike the parent drug, movement of acetaminophen metabolites requires transporters. Interaction of acetaminophen with common drug carriers has been addressed in the context of two superfamilies of transporters, solute carrier transporters (SLC) and ATP-binding cassette (ABC) transporters [Articles:17627974, 22406294, 21389119, 18789319]. ABC transporters mediate efflux of substrates from cells, while SLC transporters are responsible for uptake of substrates into cells [Articles:22013971, 16586096]. Excretion of APAP-gluc and sulfate into the bile involves ABCC2 and ABCG2 carriers found in the canalicular membrane of hepatocytes. Movement of APAP-gluc into the blood depends on ABCC3 transporter, while the sulfate metabolite relies on ABCC3 and ABCC4, both located on the sinusoidal side of liver cells [8]. In addition, ABCB1, ABCC1 and ABCC5 transporters might be involved in acetaminophen excretion in humans, as evident by changes in their expression after toxic acetaminophen ingestion [Article:17627974]. Livers from patients, who overdosed on acetaminophen, exhibit upregulation of ABCC1 and ABCC4 mRNAs and elevated protein levels of ABCB1, ABCG2, ABCC4 and ABCC5. Increased expression of efflux transporters might be an adaptive change to stop accumulation of toxic metabolites in cells and to prevent additional liver damage. Consistent with this hypothesis is increased hepatocyte proliferation and co-localization of upregulated transporters with the regions of rapidly replicating liver cells [Article:17627974]. These adaptive responses to toxic levels of acetaminophen result in acquired resistance to a repetitive insult on the liver. This phenomenon is reminiscent of autoprotection observed in experimental animals, where initial exposure to the sub-toxic doses of acetaminophen protects rodents from subsequent lethal doses of the drug [Articles:473230, 24973094]. Individual case reports suggest that human subjects can develop tolerance to repeated and high doses of acetaminophen without any liver injury [Articles:9918922, 7748624]. While the mechanism of such resistance to hepatotoxicity from acetaminophen overdose was not fully elucidated in these patients, autopotection through upregulation of efflux transporters might be responsible for the development of tolerance to chronic and lethal doses of this drug.

The SLC transporters are comprised of two gene superfamilies, the SLC22A superfamily, which contains the organic cation transporters (OCTs) and the organic anion transporters (OATs), and the SLCO superfamily, which includes the organic anion transporting polypeptides (OATPs). OATPs mostly transport large, hydrophobic organic anions, while OATs transport small and hydrophilic molecules; OCTs mediate cation movement [Article:22013971]. Using stable cell lines expressing human transporters, interaction of acetaminophen with hOATs and hOCTs was assessed [Article:12388633]. Acetaminophen inhibited organic anion uptake mediated by hOAT1 (SLC22A6), 2 (SLC22A7), 3 (SLC22A8), and 4 (SLC22A9). OCT1 (SLC22A1) and 2 (SLC22A2) did not mediate the uptake of acetaminophen, but could be inhibited by it, suggesting that acetaminophen could potentially interfere with removal of other drugs relying on these transporters [Article:12388633]. With regards to the OATP family, in vitro assays demonstrated that acetaminophen did not interact with OATP1B1 (SLCO1B1) or OATP1B3 (SLCO1B3) transporters [Article:21389119].

Drug-drug interactions

Numerous drugs have been reported to interact with acetaminophen leading to exacerbation of its toxicity [Articles:15933229, 3511825, 8470644, 2049229, 17151811, 3346037]. Several case reports suggested that epileptic patients on long-term anticonvulsant therapy exhibited increased acetaminophen-induced hepatotoxicity [Articles:3346037, 760753, 1354974, 6465717]. In most cases, chronic use of phenytoin or phenobarbital enhanced clinical features of toxicity after acetaminophen overdose [Articles:3346037, 760753, 1354974]. However, toxicity from a low therapeutic dose of acetaminophen is already potentiated if it is concurrently consumed with phenobarbital [Article:6465717]. It was suggested that epileptic patients exhibit lower bioavailability of acetaminophen due to increased first-pass metabolism of the drug [Article:760753]. Using human hepatocytes as a model for acute drug-drug interaction, phenytoin and phenobarbital were reported to inhibit acetaminophen glucuronidation [Articles:16696573, 15933229]. Each drug alone or in combination directly blocked UGT1A6, UGT1A9, and UGT2B15 when co-incubated with acetaminophen. Treatment of hepatocytes with phenytoin or phenobarbital increased acetaminophen-induced toxicity in these cells [Articles:16696573, 15933229]. This suggests that when glucuronidation is compromised, acetaminophen is directed toward other pathways, resulting in overproduction of the reactive metabolite NAPQI. It is feasible to assume that this phenomenon is responsible for enhancement of acetaminophen-induced toxicity in epileptic patients.

Many agents, including ethanol and isoniazid, induce CYP450 isozymes during their metabolism [Articles:8354023, 184555]. The antituberculosis drug isoniazid induces CYP2E1, which is crucial for acetaminophen metabolism through oxidation pathway. Co-administration of isoniazid with acetaminophen was reported to increase acetaminophen oxidation, promote GSH depletion and NAPQI formation and ultimately lead to increased hepatotoxicity [Articles:8470644, 2049229, 2240884]. CYP2E1 is also dramatically upregulated by ethanol and acetaminophen hepatotoxicity in alcoholics is well documented [Article:3511825]. Low to moderate doses of acetaminophen combined with a heavy consumption of alcohol lead to abnormal liver enzyme profile, jaundice and coagulopathy. Taken together, subjects receiving isoniazid therapy or consuming excessive amounts of alcohol should be careful with acetaminophen administration to avoid possible hepatotoxicity due to CYP2E1 induction.

Authors: Liudmila Mazaleuskaya, Katrin Sangkuhl, Caroline Thorn, Garret FitzGerald, Russ Altman, Teri Klein.
Mazaleuskaya Liudmila L, Sangkuhl Katrin, Thorn Caroline F, FitzGerald Garret A, Altman Russ B, Klein Teri E . "PharmGKB summary: pathways of acetaminophen metabolism at the therapeutic versus toxic doses" Pharmacogenetics and genomics (2015).
Therapeutic Categories:
  • Pain, anti-inflammatory and immunomodulating agents

Entities in the Pathway

Genes (21)

Drugs/Drug Classes (5)

Relationships in the Pathway

Arrow FromArrow ToControllersPMID
acetaminophen acetaminophen-glucuronide UGT1A1, UGT1A6, UGT1A9, UGT2B15 11215692, 11714888, 15180166, 17164591, 18232020, 21666065, 8494539
acetaminophen acetaminophen-sulfate SULT1A1, SULT1A3, SULT1A4, SULT1E1, SULT2A1 18232020
acetaminophen N-acetyl-p-benzoquinone imine CYP1A2, CYP2A6, CYP2D6, CYP2E1, CYP3A4 11866476, 19219744
acetaminophen-glutathione acetaminophen-cysteine 22022436, 22175791
CYP2E1 CYP2E1 ethanol, isoniazid 2240884, 8659683
N-acetyl-p-benzoquinone imine acetaminophen-glutathione GSTM1, GSTP1, GSTT1 22022436, 22175791
UGT1A6 UGT1A6 phenobarbital, phenytoin 15933229, 16696573
UGT1A9 UGT1A9 phenobarbital, phenytoin 15933229, 16696573
UGT2B15 UGT2B15 phenobarbital, phenytoin 15933229, 16696573
acetaminophen-glucuronide acetaminophen-glucuronide ABCC2, ABCC3, ABCG2 17627974, 21241071
acetaminophen-sulfate acetaminophen-sulfate ABCC2, ABCC3, ABCC4, ABCG2 17627974, 21241071

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